Parmar krupal

1. Plant Biotechnology Agrobacterium mediated gene transfer ICAR JRF, SRF, NET/ARS exam Parmar krupal

2. Agrobacterium mediated gene transfer • Agrobacterium is considered as the nature’s genetic engineer. Agrobacterium tumefaciens is a rod shaped, gram negative bacteria found in the soil that causes tumorous growth termed as crown gall disease in dicot plants. • The involvement of bacteria in this disease was established by Smith and Townsend (1907).

3. Agrobacterium contains a transfer DNA (T-DNA) located in its tumor-inducing (Ti) plasmid that is transferred into the nucleus of an infected plant cell. • The T-DNA gets incorporated into the plant genome and is subsequently transcribed.

4. Ti-plasmid • The virulent strains of A. tumefaciens harbor large plasmids (140–235 kbp) known as tumor-inducing (Ti) plasmid involving elements like T-DNA, vir region, origin of replication, region enabling conjugative transfer and • o-cat region (required for catabolism of opines).

5. T- DNA • It is a small, specific segment of the plasmid, about 24kb in size and found integrated in the plant nuclear DNA at random site. This DNA segment is flanked by right and left borders. • Genes on T-DNA • The T-DNA contains two groups of genes, which possess the ability to express in plants as follows. • Oncogenesfor synthesis of auxins and cytokinins (phytohormones). The over-production of phytohormones leads to proliferation of callus or tumour formation. • Opine synthesizing genes for the synthesis of opines (a product from amino acids and sugars secreted by the crown gall infected cells and utilized by A. tumefaciens as carbon and nitrogen sources). • Thus opines act as source of nutrient for bacterial growth, e.g. Octopine, Nopaline,Agropine.

6. Table : Functions of various T-DNA genes

7. T- DNA: Border Sequences • • T-regions are defined by direct repeats known as T-DNA border sequences (Right and Left Border i.e. RB and LB of 25 bp each). • • These are not transferred intact to the plant genome, but are involved in the transfer process. • • The RB is rather precise, but the LB can vary by about 100 nucleotides. • • Deletion of the RB repeat abolishes T-DNA transfer, but the LB seems to be non-essential. The LB repeat has little transfer activity alone. • Virulence genes (vir genes) • Virulence genes aid in the transfer of T-DNA into the host plant cell. Ti plasmid contains 35 vir genes arranged in 8 operons.

8. T DNA transfer into the plant genome

9. T DNA transfer into the plant genome i) Recognition and vir gene induction • Agrobacterium perceives signaling molecules (phenolic [acetosyrigone]compounds and sugars) released by the wounded plant cells. These signaling molecules act as chemotactic attractants for Agrobacterium. This is followed by recognition by the bacterial VirA/VirG2-component signal transduction system. • Vir A, a kinase present in the inner bacterial membrane senses these phenolics (e.g. acetosyringone), gets autophosphorylatedand then activates Vir G. VirG protein is a transcriptional activator of other vir genes and is inactive in non-phosphorylated form. The activation of Vir G thus induces the expression of the other vir genes.

10. ii) Formation of T-DNA complex Vir D1/D2 border-specific endonucleases recognize the left and right borders of T-DNA. Vir D2 induces single stranded nicks in Ti plasmid causing the release of the ss-T DNA. Vir D2 then attaches to the 5'-end of the displaced ss-T DNA forming an immature T-complex. iii) Transfer of T-DNA and integration into the plant cell The transfer of T-DNA to the plant cell composed of proteins encoded by Vir B and Vir D4 that form a conjugative pilus (T-pilus). VirD4 serves as a “linker” that helps in the interaction of the processed T-DNA/VirD2 complex with the VirB- encoded pilus. Other vir genes (Vir E2, Vir E3, VirF, Vir D5) also pass through this T-pilus to aid in the assembly of T-DNA/vir protein complex in the plant cytoplasm forming a mature T-complex. • Most VirB proteins help in the formation of the membrane channel or act as ATPases to provide energy for assembly and export processes of channel.VirB proteins, including VirB2, VirB5 and VirB7 help in the formation of the T-pilus. VirB2 is the major pilin protein that undergoes processing and cyclization. • ss-TDNA is coated with VirE2, a non-sequence specific single stranded DNA binding protein.

11. Vir D2 and Vir E2 protect the ss-T strand from nucleases inside the plant cytoplasm by attaching to the 5'end. Both VirD2 and VirE2 proteins have nuclear localization signals (NLS) which serves as pilot proteins to guide the mature T-complex to the plant nucleus. • • The efficiency of transfer is enhanced by VirC2 proteins, which recognize and bind to the overdrive enhancer element. • • Some additional proteins like importins, VIP1 and VirF may interact with the T-strand, either directly or indirectly, to form larger T-complexes in the plant cell. • Inside the nucleus, ss-TDNA is converted into ds-TDNA which gets integrated into the plant genome via process called illegitimate recombination.

13. Disarmed Ti-plasmid • For efficient plant regeneration, vectors with disarmed T-DNA are used by making it non-oncogenic by deleting all of its oncogenes. The foreign DNA is inserted between the RB and LB and then integrated into the plant genome without causing tumors. Fig Structure of the Ti-plasmid pGV3850 with disarmed T-DNA.

14. Co- integrate vectors • Vectors that recombine via DNA homology into resident Ti plasmid are referred as integrative or co integrate vectors. • In this the disarmed Ti vector is covalently linked to donor vector with gene of interest T DNA border sequence present in a A. tumefaciens strain to act as one unit. • In this much of wild type T DNA especially hormone biosynthetic genes and not border genes is replaced with a segment of DNA common to many E. Coli clonning vector. • Homology between E.coli plasmid based segment and of the modified T DNA and the donor vector with identical sequences provides a site of recombination to occur.

15. Donor vector Fig Construction of a Co-integrate vector (foreign gene cloned into an appropriate plasmid is integrated with a disarmed Ti-plasmid through homologous recombination).

16. Binary vector Binary vector was developed by Hoekmaet al (1983) and Bevan in (1984). • It utilizes the vir genes of the Ti-plasmid and can act on any T-DNA sequence present in the same cell. • Binary vector contains transfer apparatus (the vir genes) and the disarmed T-DNA containing the transgene on separate plasmids Advantages of Binary vector • Small size due to the absence of border sequences needed to define T-DNA region and vir region. • Ease of manipulation pBIN19- one of the first binary vectors developed in 1980s and was widely used

17. Fig binary vector system (a) A plasmid containing vir region but no T-DNA, therefore no T-DNA transfer takes place in plant genome. (b) Another plasmid containing T-DNA with Right border (RB) and Left border (LB) but no vir genes. Vir function is supplied by former plasmid.